The exhaustion of fossil fuels is an eminent concern facing the industrialized world. With an ever-growing energy demand, the need for sustainable alternative renewable fuels is greatly needed. In addition, there is the negative impact linking increased carbon emissions with the burning of petroleum products to global warming.
Renewable liquid hydrocarbon-based fuels such as biodiesel are a potential candidate to resolve these energy concerns due to its high energy density and maintaining a neutral carbon footprint. The use of biodiesel maintains a balanced carbon dioxide cycle, as the carbon is recycled through renewable biological materials without releasing additional carbon into the atmosphere as petroleum based fossil fuels. Biodiesel is a monoalkyl ester of fatty acids and is nontoxic; it does not contain sulfur, aromatic hydrocarbons, or other toxic side-products linked to pollution found in petroleum diesel. It can also act as a lubricant in diesel engines thus reducing wear on engine parts. Pure biodiesel or mixtures of biodiesel and petroleum-based diesel can be used in unmodified diesel engines without changing the current infrastructure to distribute it.
Current manufacturing methods of biodiesel use vegetable oils for production; the broad use of it is hindered by the extensive acreage required for sufficient production of oilseed crops from such sources as palm oil in south East Asia, rapeseed in Europe, and soybean in North America (Kalscheuer et al., 2006. Microbiology 152:2529-36). Vegetable oil is a triacylglycerol (TAG) neutral lipid that can be burned in modified diesel engines, however, the viscosity is high and it doesn't burn completely thus leaving an oily residue. The current method for biodiesel production is alcoholysis of vegetable oil, which uses methanol to transesterify the glycerol group away from the fatty acid groups forming a fatty acid methylester (FAME). Additives can be combined with FAMEs to decrease viscosity and using desaturated fatty acids can make biodiesel less viscous at lower temperatures where saturated FAMEs would gel (using desaturated oils such as oleic acid).
Another biodiesel source being investigated is jojoba wax oil, which is composed of a long-chain fatty alcohol that is transesterified with methanol (Canoira et al., 2006. Biomass and Bioenergy 30:76-81; Verschuren and Nugteren. 1989. Food Chem Toxicol 27:45-8; Wisniak, 1977. Prog Chem Fats Other Lipids 15:167-218). However, jojoba oil is more expensive to harvest and process compared to vegetable oils (Kalscheuer et al., 2006. Appl Environ Microbiol 72:1373-9).
Enzymatic attempts at making biodiesel have also been investigated using lipases (Akoh et al., 2007. J Agric Food Chem 55:8995-9005). Lipases are alpha-beta hydrolases that break down neutral lipids such as TAGs. However, if lipases are in a solvent system (i.e. toulene), lipases will perform a transesterification reaction exchanging the glycerol for methyl alcohol functional groups. Lipases can either be immobilized or expressed in recombinant cells that are imbedded onto a matrix and are emulsified with vegetable oils and methanol to perform the transesterification synthesis.
However, current problems with the transesterification process include excessive accumulations of glycerol byproducts and the continued use of toxic, petroleum-derived methanol. The methanol used for FAME synthesis is produced from natural gas; therefore with the use of fossil fuel petroleum component, FAME-based biodiesel is not truly a renewable biofuel. In addition, the transesterification process and subsequent purification steps are more expensive and energy consuming, thus reducing the energy yield and increasing the costs to make biodiesel (Kalscheuer et al., 2006. Microbiology 152:2529-36).
The first microbial wax ester synthase/acyltransferase (WS/DGAT) was characterized from Acinetobacter baylyi ADP1 and showed substrate promiscuity (Stoveken et al., 2005. J Bacteriol 187:1369-76). WS/DGAT has also been heterologously expressed in microbial cell lines such as Pseudomonas citronellolis (Kalscheuer and Steinbüchel. 2003. J Biol Chem 278:8075-82) creating wax esters and fatty acid butyl esters (FABEs), which have also been synthesized in recombinant Escherichia coli (Kalscheuer et al., 2006. Microbiology 152:2529-36). WS/DGAT has also been expressed in eukaryotic hosts creating TAGs, fatty acid ethyl esters (FAEEs) and fatty acid isoamyl esters (FAIES) in recombinant Saccharomyces cerevisiae (Kalscheuer et al., 2004. Appl Environ Microbiol 70:7119-25). Furthermore, wax diesters and wax thioesters have been synthesized in mutant A. baylyi strain ADP1 acr1VKm (Kalscheuer and Steinbüchel. 2003. J Biol Chem 278:8075-82; Uthoff et al., 2005. Appl Environ Microbiol 71:790-6). In 2006, Kalscheuer et al created a FAEE termed ‘microdiesel’ where ethanol-producing genes from Zymomonas mobilis (Carvalhal et al., 1996. Revista De Microbiologia 27:263-267) and the acyltransferase, WS/DGAT enzyme from Acinetobacter baylyi ADP1, were heterologously expressed in E. coli strain harboring a plasmid with all three genes. FAME and FAEE are similar in their chemical and physical combustion properties (Antoni et al., 2007. Appl Microbiol Biotechnol 77:23-35; Kalscheuer et al., 2006. Microbiology 152:2529-36).
However, E. coli is often not a suitable host for the creation of the biodiesel. The FAEEs yields were below the requirements of a sustainable industrial process; therefore fatty acids had to be supplemented to the recombinant E. coli strain in the form of sodium oleate (Kalscheuer et al., 2006. Microbiology 152:2529-36). De novo fatty-acid biosynthesis in E. coli does not provide ample acyl substrates for the Acinetobacter WS/DGAT-mediated FAEE synthesis, which indicates that this microbe may not be the ideal host for biodiesel production.
Furthermore, the expression host determines the types of acyl esters synthesized by Acinetobacter WS/DGAT based upon the biochemical/physiological background and the access to substrates (i.e. ethanol) made available either by natural metabolism, medium supplementation, or genetic engineering.
Neutral lipid biosynthesis is ubiquitous in nature and occurs in animals, plants, and microbes. Microorganisms have been reported to synthesize TAGs (Wältermann et al., 2005. Mol. Microbiol. 55:750-763), polyhydroxyalkonates (PHAs) (Steinbüchel, 2001. Macromol. Biosci. 1:1-24), and wax esters (WEs) (Wältermann and Steinbüchel. 2005. J. Bacteriol. 187:3607-3619). Neutral lipids accumulate as inclusion bodies within the microbial cell, and their purpose is to serve as carbon and energy storage under growth-limiting conditions. PHAs are composed of aliphatic monomeric unit polyesters, which are the most abundant class of neutral lipids in microbial species (Steinbüchel, 2001. Macromol. Biosci. 1:1-24). It is believed that neutral lipid inclusion bodies not only serve as an energy storage but also remove fatty acids that may cause damage to the bacterial cell membrane (Alvarez et al., 2002. Microbiology 148:1407-1412). Until recently, only microbial PHA biosynthesis has been investigated, and their biochemistry and metabolism has been well described (Steinbüchel, 2001. Macromol. Biosci. 1:1-24). The enzymes involved in microbial TAG biosynthesis and WE have only very recently been identified (Daniel et al., 2004. J. Bacteriol. 186:5017-5030; Kalscheuer and Steinbüchel. 2003. J. Biol. Chem. 278:8075-8082; Wältermann et al., 2000. Microbiology 146:1143-1149; Wältermann et al., 2007. Biochemie 89:230-242).
Microbial WEs have been found in Mycobacterium (Daniel et al., 2004. J. Bacteriol. 186:5017-5030), Rhodococcus (Alvarez et al., 2002. Microbiology 148:1407-1412), Acinetobacter (Alvarez et al., 1997. Appl. Microbiol. Biotechnol. 47:132-139), and Marinobacter (Rontani et al., 2003. 69:4167-4176; Rontani et al., 1999. Appl. Environ. Microbiol. 65:221-230) strains that grow in environments where a carbon source (such as petroleum hydrocarbons [Ishige et al., 2003. Curr. Opin. Microbiol. 6:244-250] and gluconate [Kalscheuer and Steinbüchel. 2003. J. Biol. Chem. 278:8075-8082]) may be abundant relative to other nutrients such as phosphorous and nitrogen. Acyl WEs are synthesized from long-chain fatty alcohol and fatty acyl-coenzyme A (CoA) substrates. Another class of WEs is the isoprenoid WEs that are made from branched, long-chained isoprenoyl alcohol and isoprene fatty acid substrates. Isoprenoid WEs have been identified as a way to provide energy storage in Marinobacter species (Rontani et al., 2003. 69:4167-4176; Rontani et al., 1997. Appl. Environ. Microbiol. 63:636-643; Rontani et al., 1999. Appl. Environ. Microbiol. 65:221-230). Marinobacter species grow in marine sediment materials where there is an abundance of recalcitrant acyclic isoprenoid alcohols such as farnesol and phytol, which are derived from (bacterio) chlorophyll molecules (Rontani et al., 1997. Appl. Environ. Microbiol. 63:636-643; Rontani et al., 1999. Appl. Environ. Microbiol. 65:221-230).
A microbial WS/DGAT capable of catalyzing WE synthesis and, to a lesser degree, TAG synthesis was identified in the gamma proteobacterium Acinetobacter baylyi ADP1 (Kalscheuer and Steinbüchel. 2003. J. Biol. Chem. 278:8075-8082; Stöveken et al., 2005. J. Bacteriol. 187:1369-1376; Uthoff et al., 2005. Appl. Environ. Microbiol. 71:790-796). The A. baylyi ADP1 WS/DGAT contains the catalytic motif HHXXXDG (SEQ ID NO:21) that is involved in the acyltransferase reaction (Pfam domain PF00668) (Kalscheuer and Steinbüchel. 2003. J. Biol. Chem. 278:8075-8082). This motif has been found in numerous sequenced genomes of microbial strains that are known to make WEs and/or TAGs and is also found in the condensation domain of some nonribosomal peptide synthetase (NRPS) modules. Mutations within this domain have been shown to abolish NRPS activity (Stachelhaus et al., 1998. J. Biol. Chem. 273:22773-22781; Wältermann et al., 2007. Biochemie 89:230-242). Two WS/DGAT homologues from the marine hydrocarbonoclastic bacterium Alcanivorax borkumensis (Kalscheuer et al., 2007. J. Bacteriol. 189:918-928) have been reported.
The gamma proteobacteria Marinobacter hydrocarbonoclasticus DSM 8798 has been shown to synthesize an isoprenoid WE when grown on phytol as the sole carbon source and under nitrogen-limiting conditions (Rontani et al., 2003. 69:4167-4176; Rontani et al., 1999. Appl. Environ. Microbiol. 65:221-230). It has been proposed that exogenous phytol is transported into the cell, where it is converted into an intermediate aldehyde (phytenal) that is then further oxidized into the isoprenic fatty acid phytenic acid, which may be hydrogenated into phytanic acid (Rontani et al., 1999. Appl. Environ. Microbiol. 65:221-230). Phytanic acid is then esterified with phytol to form an isoprenoid WE.